【作者】 周涛；

【导师】 王玉琦； 符伟国； Omaida Velazquez；

【作者基本信息】 复旦大学 ， 血管外科， 2009， 博士

【摘要】 第一部分内皮祖细胞的分离培养和鉴定目的:利用实验室已建立的外周血内皮祖细胞(endothelial progenitor cells,EPCs)分离培养方案,建立EPCs的培养体系并进行细胞表型和功能的检测与鉴定,为后续的实验研究提供细胞来源。方法:采集新西兰大白兔静脉外周血20ml,以密度梯度离心法获得单个核细胞,将细胞放入涂有0.1%纤连蛋白的培养皿中,在加有20%胎牛血清(FBS)和EGM-2 SingleQuots的内皮细胞基础培养基-2(EBM-2)中培养。于培养第7d应用免疫荧光检测细胞标志CD133,CD34,vWF,CD45抗原的表达;利用DiI-acLDL和FITC-UEA-1吸收试验鉴定内皮功能。结果:单个核细胞在内皮条件下培养,48h后逐渐贴壁,第3d细胞呈梭形或不规则形,并逐渐变大,第7d梭形细胞增多,部分视野观察到梭形细胞首尾相连形成条索样结构。荧光显微镜观察CD133,CD34,vWF阳性率分别为83.11±3.46%,82.87±4.66%,77.45±6.71%,CD45阴性。并且,有＞90%的细胞吸收DiI-ac LDL和FITC-UEA-1。结论:应用本研究方法从外周血途径分离、培养和扩增获得EPC,具有内皮祖细胞的特征和功能。第二部分基质细胞衍生因子-1对内皮祖细胞的体外作用目的:制备本实验室已冻存的病毒基因载体转导细胞NIH3T3/SDF-1和NIH3T3/LacZ细胞并检测细胞功能,利用其分泌基质细胞衍生因子-1(SDF-1)的特点研究对体外培养、扩增的EPC的影响。方法:解冻NIH3T3细胞通过逆转录病毒转导SDF-1α基因和LacZ基因后制作能表达SDF-1的NIH3T3细胞(NIH3T3/SDF-1)和表达LacZ的NIH3T3/LacZ细胞。用ELASA法测定NIH3T3细胞,NIH3T3/SDF-1细胞在体外SDF-1表达情况,及存在EPO(10IU/ml)是对细胞表达SDF-1的影响,β-Gal染色法鉴定NIH3T3/LacZ细胞。用改良Boyden小室法检测NIH3T3/SDF-1细胞表达的SDF-1对内皮祖细胞EPCs体外迁移作用,DAPI染色法检测SDF-1对EPCs凋亡影响。结果:冻存细胞可顺利解冻,培养细胞存活细胞率≥90%。转导入SDF-1表达载体的NIH 3T3/SDF-1细胞24小时可产生147±6 ng SDF-1/10~6细胞,转导入LacZ表达载体的NIH3T3/LacZ细胞可表达LacZ,细胞β-Gal染色阳性,为蓝色。EPO的存在并不能直接影响NIH3T3和NIH3T3/SDF-1细胞中SDF-1的表达。当EPCs与NIH3T3/SDF-1细胞共培养时同单独NIH3T3细胞共培养时相比迁移较多,(分别为31±9和13±4迁移细胞,P＜0.05)。并且,当在L-NMMA存在时,EPCs和NIH 3T3/SDF-1细胞共培养后迁移细胞明显减少为16±4(P＜0.05)。而与NIH 3T3/SDF-1细胞共培养的EPCs中凋亡发生率与同NIH 3T3细胞共培养的EPCs中凋亡发生率相比较低(分别为17±4%,30±7%P＜0.05)。L-NMMA的存在并不明显影响与NIH 3T3/SDF-1细胞共培养的EPCs中凋亡发生率(18±5%P＞0.05)。结论:体外迁移实验表明EPC可在NIH3T3/SDF-1细胞分泌SDF-1的趋化作用下迁移,SDF-1可抑制EPCs凋亡。SDF-1迁移效应是通过一氧化氮(NO)途径,但NO没有参加SDF-1抑制EPC凋亡的过程。EPO并不影响NIH3T3/SDF-1细胞SDF-1的表达。本研究为下一步研究SDF-1在体内趋化EPC以及促血管新生的研究打下了必要的基础。第三部分促红细胞生成素联合基质细胞衍生因子对小鼠缺血下肢血管新生的作用目的:促红细胞生成素(EPO)可促进骨髓单核细胞动员到外周血中,基质细胞衍生因子1(SDF-1)可促进从骨髓动员后的祖细胞的在缺血组织中的归巢和血管新生。我们假设联合应用EPO和SDF-1会大大增加促进缺血组织中血管新生的作用。方法:将C57BL/6J小鼠股动脉和股静脉切除后制作小鼠下肢缺血模型,分为EPO或SDF-1单独处理组和联合应用组。SDF-1应用方法为在缺血肌肉中立即注射NIH3T3/SDF-1细胞(10~6)以局部表达SDF-1,EPO应用方法为术后促红细胞生成素(EPO 1000IU/kg/day)每天腹腔注射,连续3天。小鼠缺血模型下肢血流灌注情况用激光多普勒扫描测定。下肢缺血手术后21天处死小鼠并用免疫组化和Western印迹方法分析缺血肌肉组织中细胞SDF-1表达,免疫方法检测缺血组织内CD34~+细胞,碱性磷酸酶染色方法评估毛细血管密度,原位凋亡测定试剂盒检测肌肉细胞的凋亡。全自动血液分析仪检测外周血液学参数以明确EPO对外周血细胞的影响。结果:免疫组化和Wwestern印迹分析表明注射的NIH3T3/SDF-1细胞在缺血组织内生存并表达SDF-1。用SDF-1或EPO单独处理3周后缺血/非缺血肢体的血流灌注比分别增加到0.75±0.06和0.63±0.03,同注射生理盐水对照组相比增加明显(0.50±0.02,P＜0.05)。而联合应用SDF-1和EPO组小鼠缺血下肢血流灌注比增加更明显(0.89±0.08同单独一种处理相比P＜0.05)。在SDF-1和EPO处理组,CD34~+细胞增加,毛细血管密度增加和肌肉细胞凋亡减少均比其他组相比有意义(同其他组相比P＜0.05)。外周血红细胞数目和红细胞压积无明显增加。结论:在小鼠下肢缺血模型中联合应用EPO增强EPCs动员和增加SDF-1调节的内皮祖细胞(EPCs)归巢可促进血管新生和增加血流灌注。同时,短期应用EPO并不增加红细胞数量和红细胞压积。更多还原

【Abstract】 PartⅠIsolation and characterization of endothelial progenitor cellsObjective:To establish the culturing system of EPCs according to the former established protocols of our lab,and provide cell source for following experiments.Method:Mononuclear cells(4×10~6) were isolated by density gradient centrifugation from rabbit peripheral blood(20 ml) plated on culture dishes coated with fibronectin (0.1%),and cultured in endothelial cell basal medium-2(EBM-2) supplemented with 20%fetal bovine serum(FBS) and Clonetics EGM-2 SingleQuots.The EPC were characterized by acetylated low density lipoprotein(DiI-acLDL) uptake,lectin binding(FITC-UEA-1),and immunochemical staining for CD34,CD133,CD45,von Willebrand factor(vWF),at 7 days after onset of culture.Results:Attached spindle-like cells appeared after 3 days culture and became more and larger at 7 days of culture.Cord-like structure was observed.The positive rates of CD133,CD34,andⅧfactor of ex vivo expanded EPC at 7 days of culture were 83.11±3.46%,82.87±4.66%and 77.45±6.71%,respectively.CD45 is negative.The cells had the capacity to incorporate DiI-acLDL and FITC-UEA-1(＞90%).Conclusion:EPCs have been shown to be present in circulation,and could be isolated and cultured from peripheral blood MNCs.PartⅡStromal cell-derived factor-1 effects on endothelial progenitor cell in vitroObjective:To prepare NIH 3T3 transduced with the SDF-1αretroviral vector(NIH 3T3/SDF-1) and with LacZ retroviaral vector(NIH 3T3/LacZ) and investigate the effect of stromal cell-derived factor-1(SDF-1) secreted by NIH 3T3/SDF-1 on migration and apoptosis of ex vivo expanded EPCs. Methods:Thaw the freezing NIH 3T3/SDF-1 cells and the SDF-1 concentration in the supernatant of cultured cells with or without EPO was measured with enzyme-linked immunosorbent assay(ELISA).Thaw the freezing NIH 3T3/LacZ cells and was conformed by theβ-Gal staining.Amodified Boyden chamber assay was used to determine the effect of secreted SDF-1 on EPC migration.Apoptosis of EPCs under influence of SDF-1 were investigated by stained with DAPI with or without L-NMMA.Results:The freezing cells were thawed successfully and cells viability≥90%.NIH 3T3/SDF-1 cells produced 147±6 ng of SDF-1 per 10~6 cells in 24 h and EPO can not influence the expression of SDF-1 in NIH 3T3/SDF-1 cells.NIH3T3/LacZ cells were conformed by positiveβ-Gal staining.EPCs migrated more when they were co-cultured with NIH 3T3/SDF-1 than with unmodified NIH 3T3 cells(31±9 versus 13±4 migrated cells,respectively;P＜0.05).The migrated cells were significantly reduced to 16±4 when EPCs were cocultured with NIH 3T3/SDF-1 in the presence of L-NMMA(P＜0.05).Apoptosis was found in 30±7%of nutrient deprived EPCs co-cultured with NIH 3T3 cells.EPCs co-cultured with NIH 3T3/SDF-1 demonstrated a significant reduction in apoptosis(17±4%,P＜0.05) L-NMMA did not significantly affect apoptosis in EPCs co-cultured with NIH 3T3/SDF-1 cells(18±5%,P＞0.05).Conclusion:NIH3T3/SDF-1 cells can secrete SDF-1 to induce EPC migration in vitro through NO pathway.Apoptosis of EPCs can be reduced by SDF-1 secreated by NIH3T3/SDF-1cells and NO is not involved in the inhibition of EPC apoptosis.PartⅢErythropoietin with Stromal cell-derived factor-1 in therapeutic nevascularization of endothelial progenitor cells in ischemia limbObjective:Erythropoietin(EPO) mobilizes bone marrow mononuclear cells into the peripheral circulation.Stromal cell-derived factor-1(SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue.We hypothesize that SDF-1 will boost the pro-angiogenic effect of EPO.Methods:NIH 3T3/SDF-1(10~6 cells) cells were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. EPO(1000IU/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system.Mice were sacrificed 21 days after surgery.Immunostaining and Western blot assay of the tissue lysates to confonfirm the cells in ischemia tissues and expressed SDF-1. Capillary density in the ischemia tissue were assessed with alkaline phosphatase staining,and the apoptosis of muscle cells was viewed using an in situ cell death detection kit.Results:The perfusion ratio of ischemic/non-ischemic limb increased to 0.75±0.06 and 0.63±0.03 with the treatment of either SDF-1 or EPO only,respectively,3 weeks after surgery,which was significantly higher than the saline-injected control group (0.50±0.02,P＜0.05).Combined treatment with both SDF-1 and EPO resulted in an even better perfusion ratio of 0.89±0.08(P＜0.05 versus the single treatment groups). CD34~+ cells were detected with immunostaining,and Western blot assay showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1.More CD34~+ cells, increased capillary density,and less apoptotic muscle cells were found in both EPO and SDF-1 treated group(P＜0.05 versus other groups).Conclusion:Combination of EPO-mediated progenitor cell mobilization and SDF-1-mediated homing of EPCs promotes neovascularization in the ischemic limb and increases the recovery of blood perfusion without increasing the number of red blood cells and hematocrit.更多还原